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同志社大学リエゾンオフィス https://kikou.doshisha.ac.jp/T E L：0774-65-6223E-mail：[email protected]
Doshisha University
有毒ガスでありながら生体内シグナルガスとしても注目される一酸化炭素の生体内除去およびデリバリーツール
同志社大学理工学部機能分子・生命化学科北岸 宏亮 Hiroaki KITAGISHI
一酸化炭素
有毒ガス，火災の死亡原因，排気ガス，タバコ，練炭自殺…etc.
生理活性ガス，ヘムタンパク質へのシグナル，抗炎症作用，臓器保存効果…etc.
有毒ガス ＝ 生体内で作用する＝ 微量は生物に必要
CO
内因性の一酸化炭素の産生について
N
N N
N
FeIII
COOHHOOC
N
NH HN
HN
COOHHOOC
O O
+ Fe２+
heme(Fe3+ hemin)
biliverdin
heme oxygenase
heme metabolism metabolic products
CO
CO-ヘモグロビンとして循環
細胞内シグナルガスとして機能
NS S
Fe(II)
NS S
Fe(II)
O2
NS S
Fe(II)
CO
hemoCD
oxy-hemoCD CO-hemoCD
hemoCD
SO3-
S S
N
N
N
N
NSO3
--O3S
-O3S
FeII
3 3
P50 (O2) / Torr P50 (CO) / Torr
Hb (T-state) 22 -
Hb (R-state) 0.22 0.0013
hemoCD 10 0.000015
K. Kano, H. Kitagishi et al Inorg. Chem. 2006, 45, 4448–4460.
HemoCD is a useful CO receptor in aqueous media.
An Hb/Mb functional model
SS
N
O
OHO
H3CO
OCH3
OO
O
OCH3
H3CO
H3CO
OO
OCH3
OCH3
H3CO O
O
H3CO
OCH3
H3CO
O
O
H3CO
OCH3
OCH3
O
OH3CO
OCH3
OCH3O
OCH3
H3CO
O
OOHOCH3
H3CO
OO
O
H3COOCH3
OCH3
O O
H3CO
H3COOCH3
O
O
OCH3
H3CO
OCH3
O
O
OCH3H3CO
OCH3
O
O OCH3
H3CO
H3CO
O
OCH3
OCH3
N N
N N
SO3–
SO3–
SO3–
–O3S FeII+
FeIITPPSPy3CD
一酸化炭素の捕捉物質hemoCDについて
NS S
Fe(II)
O2
oxy-hemoCD
NS S
Fe(II)
CO
CO-hemoCD (in urine)
A CO-removal agentin the living organisms
Endogenous CO-depleted state
Pseudo-knockdownof CO in mice
Administration of hemoCD
Angew. Chem. Int. Ed. 2010, 49, 1312–1315.
動物体内のCOを尿中へと強制的に追い出す
内在性COを欠乏した疑ノックダウンマウス
N
N N
N
FeIII
COOHHOOC
N
NH HN
HN
COOHHOOC
O O
+ Fe2+ + COHO-1
Knockout/knockdown cannot be available for CO.
N
N N
N
FeIII
COOHHOOC
CO
Knockout/knockdown methods are available.H2S
NO
COの生理機能には不明な点が多く，注目ターゲット
一酸化炭素の捕捉物質hemoCDについて
同志社大学リエゾンオフィス https://kikou.doshisha.ac.jp/T E L：0774-65-6223E-mail：[email protected]
Doshisha University
NS S
Fe(II)
O2
oxy-hemoCD
NS S
Fe(II)
CO
CO-hemoCD (in urine)
A CO-removal agentin the living organisms
Endogenous CO-depleted state
Pseudo-knockdownof CO in mice
Administration of hemoCD
Angew. Chem. Int. Ed. 2010, 49, 1312–1315.
動物体内のCOを尿中へと強制的に追い出す
内在性COを欠乏した疑ノックダウンマウス
12
9
6
3
0
PBS
Fb-hemoCD
30 min
90 min
Oxy-hemoCD
Hmox-1/b-actin
180 m
in
**
**
*
N
N N
N
HOOC COOH
Fe
N
NH HN
HN
HOOC COOH
O O
Fe3+ COHO-1
hemin
oxy$hemoCD
CO$hemoCD
induc&on
*
**
**
Fe2+
HO-1
induction
The expression of HO-1 strongly increased in the hemoCD-treated mice.
CO恒常性維持のためのフィードバックシステムの発見！
Removal of endogenous CO.
J. Am. Chem. Soc. 2016, 138, 5417-5425.
NS S
Fe(II)
O2
HepG2 HeLa RAW264 NIH3T3 CHO SH-SY5Y
MC
O /
pm
ol p
er
10
6 c
ells 300
100
0
200
cancer cells normal cells
細胞内にある内在性COの定量に成功！
1 x 106 cells
adhered on the dish
removal of medium
oxy-hemoCD
collection of the cells
cell suspension
1) cell count2) sonication3) filtration
filtrate in cuvatte
CO
/p
mo
lpe
r1
06ce
lls
350 450 550 650Wavelength / nm
Ab
so
rba
nce
spectrum a (filtrate)
spectrum b (a + CO)
spectrum c (b + Na2S2O4)
**
in PBS
J. Am. Chem. Soc. 2017, 139, 5984.
HemoCDは微量COの検出試薬としても使える
Measurement for biological CO
外来性COの臓器分布計測新生児黄疸の早期診断法への応用etc.
１) ガスセンサー/ガスクロマトグラフィー法ヒトの場合：呼気のCOを測定し，血中CO量を推定する血液/組織：COの結合したヘムを酸化し，遊離COの気相分析を行う
２) 赤外レーザー分光法細胞内COを感度良くリアルタイム計測可能だが，特殊装置が必要(Y. Morimoto, et al., Am. J. Physiol. 2001, 280, H483)
３) ケミカルプローブの利用CO選択的な蛍光プローブ (BODIPY-Pd錯体) 定量性には乏しい(C. J. Chang, et al., J. Am. Chem. Soc. 2012, 134, 15668)
HemoCD アッセイ
Hbなどの生体内のあらゆるヘムタンパク質からCOを奪い，吸光度測定により簡単かつ正確にCOを定量可能
NEW!
内在性COの欠乏は，体内時計のリズムを乱す
NS
N
N
N
N
SO3–
SO3––O3S FeII
–O3S
E-box clock genes
Per Cry
PER CRY
BMAL1degradation
1 day
Per, Cry
Circadian time
CLOCKor NPAS2
Sci. Rep. 2018, 8, 11996 (12 pages).
0.3
0.2
0.1
0control 30 min 90 min
CO
-Hb / %
180 min
Time after the administration
*
*
*
The CO-Hb content (%) in the blood
was measured by gas chromatography.
CO-ヘモグロビンの量がほとんどゼロになった
すぐに定常値へと回復した。すなわちCOの産生が亢進された？
0.2%
0.03%
(1 mM, 0.15 mL)
NS S
Fe(II)
O2
oxy-hemoCD
blood sampling
0.2%
J. Am. Chem. Soc. 2016, 138, 5417-5425.
血中COヘモグロビンの定量
同志社大学理工学部機能分子・生命化学科北岸 宏亮 Hiroaki KITAGISHI
有毒ガスでありながら生体内シグナルガスとしても注目される一酸化炭素の生体内除去およびデリバリーツール
一酸化炭素の捕捉物質hemoCDについて
Measurement for biological CO
同志社大学リエゾンオフィス https://kikou.doshisha.ac.jp/T E L：0774-65-6223E-mail：[email protected]
Doshisha University
CORM -401
R. Motterlini
COの継続投与は，体重増加を効果的に抑制する＝ダイエットピル？
3insight.jci.org https://doi.org/10.1172/jci.insight.123485
RESEARCH ARTI CLE
to basal levels by CORM -401 in the HFD + CORM -P group but not in HFD + CORM -T group. In contrast,
mRNA expression of PPar-α, Pgc1-α, and Cpt1 was decreased by HFD but remained unchanged after CORM -
401 treatment (data not shown). Significant remodeling in other fat pads, particularly in brown adipose tissue
(BAT), was evident in the reduction of weight and lipid content in both groups administered with CORM -
401 (Supplemental Figure 3, A–E). Similarly to that in eWAT, mRNA expression of the metabolic genes
PPar-γ, Vegf, adiponectin, Pgc1-α, and Ucp1 was decreased in BAT of HFD-fed obese mice and fully or partially
restored in the HFD + CORM -P group (Supplemental Figure 3G). Conversely, gene expression of metabolic
markers in subcutaneous adipose tissue was not affected by CORM -401 (Supplemental Figure 3F). Despite
the fact that HFD did not induce a high-grade inflammation in our model, CORM -401 still modulated local
inflammation in obese mice. First, the increased macrophage infiltration induced by HFD was virtually abol-
ished by CORM -401 administration (Figure 2, F and G), although the induction of Ccl2 was unchanged by
the compound (Figure 2H). Second, CORM -401 decreased Hmox-1 expression induced by HFD (Figure 2I )
Figure 1. CORM-401 reduces body weight gain and improves insulin resistance in high-fat diet–induced obesity. Mice received a standard diet (SD) or
high-fat diet (HFD) for 14 weeks. CORM-401 (30 mg/kg) was given by oral gavage starting either at the beginning of (HFD + CORM-T) or after 6 weeks of
(HFD + CORM-P) HFD. Carbonmonoxy hemoglobin (COHb) was measured after oral gavage with CORM-401 (A). Body weight (BW ) was measured every
week (B) and BW gain was calculated as the percentage of the basal weight (C). A glucose tolerance test (GTT) was performed at week 13 (D), and data
represent the area under the curve (GTT AUC) (E). An insulin tolerance test (ITT) was performed at week 14 (F), and data represent the area under the curve
(ITT AUC) (G). Fasting total cholesterol (H), fasting triglycerides (I), fasting glucose (J), fasting insulin (K), calculated HOMA-IR (L), and fasting leptin (M)
were measured after 14 weeks in all groups examined. Values represent the mean ± SEM. n = 4–6 mice per group (A); n = 8–10 mice per group (B–M). *P <
0.05 vs. control group (SD), #P < 0.05 vs. HFD group, values not designated with symbols are not statistically di f erent, Student’s t test or 1-way or 2-way
ANOVA with Fisher multiple comparison test.
JCI Insight, 3, e123485 (2018).
水溶性，3CO/molecule放出
Cellular uptake of CORM-401
HepG2 cells
CORM-401 (50 µM in D-MEM/10% or 1% FBS)Incubation for 2 h
disrupt cells by sonication
measurement byUV-vis spectrum
wash by PBS
5 µM oxy-hemoCD1
Figure 2. Quantification of endogenous CO in cells after the addition
of 50 µM CORM-401 by hemoCD. Cells were incubated in D-MEM/10% or 1% FBS.
血清を減らすことで、細胞へのCO送達量が2.1倍増加した
細胞導入率10% FBS : 0.4%
1% FBS : 0.8%
投与量に対して非常に少ない
HON
CH3O
S
S
MnI
COCO
COCO
CORM-401
血清の成分はCORMの細胞導入を阻害Silva, T. S., et al., J. Am. Chem. Soc. 2011, 133, 1192-1195.
0
1
2
3
4
5
6
CO
-hem
oC
D1 / c
ell (x 1
0-1
6 m
ol)
7
without CORM CORM+10% FBS
CORM+1% FBS
8
2.3 x 10-16 mol/cell
2.1 x 10-16 mol/cell
1 x 106 cell / mL
Removal of
medium
D-10 1 mL
CORMs 50 µM
Incubation 2 h Removal of
medium
PBS 1 mL
Oxy-hemoCD
Collection of cells
Cell count
Sonication
Filtration
Measured by UV-vis
同志社大学理工学部機能分子・生命化学科北岸 宏亮 Hiroaki KITAGISHI
有毒ガスでありながら生体内シグナルガスとしても注目される一酸化炭素の生体内除去およびデリバリーツール
大量合成できるCORM401から，簡便で後処理がほとんどなくメチルエステル化に成功した
従来のCORM401よりも5倍以上の高い胞内取り込み効率を示した
COの研究をするための研究用試薬としての需要は高い
CORM401-E
北岸宏亮，高山実花子，R. Motterlini, 特許出願中
一酸化炭素徐放試薬CORMについて
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